Eimeria antigenic composition which elicits antibodies against avian coccidiosis

ABSTRACT

This invention relates to novel recombinant antigenic proteins of avian coccidiosis, and fragments thereof containing antigenic determinants, and to the genes that encode the antigenic peptides. This invention also relates to vaccines made using the novel antigenic proteins of avian coccidiosis and to methods of immunizing chickens against avian coccidia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.08/148,432, filed Nov. 8, 1993, now U.S. Pat. No. 5,482,709, which is adivisional application of application Ser. No. 07/581,693, filed Sep.12, 1990, now U.S. Pat. No. 5,273,901, which is continuation-in-part ofPCT/US89/02918, filed Jul. 5, 1989, which is a continuation-in-part ofU.S. patent application Ser. No. 07/215,162, filed Jul. 5, 1988 nowabandoned; which is a continuation-in-part of U.S. patent applicationSer. No. 06/746,520, filed Jun. 19, 1985, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 06/627,811,filed Jul. 5, 1984, now abandoned.

FIELD OF THE INVENTION

This invention is in the field of avian coccidiosis and is directed torecombinant antigenic proteins of avian coccidia and to the genes thatencode the proteins. These antigenic proteins may be used in a vaccineagainst avian coccidia.

BACKGROUND OF THE INVENTION

Coccidiosis is a disease of both invertebrates and vertebrates,including man, caused by intracellular parasitic protozoa whichgenerally invade the epithelial cells lining the alimentary tract andthe cells of associated glands. The crowded conditions under which manydomestic animals are raised have contributed to increased incidence ofthe disease. Virtually every domestic animal is susceptible toinfection, and distribution of the parasite is world-wide. Coccidiosisis therefore the cause of significant economic loss throughout theworld.

The poultry industry suffers particularly severe losses, withcoccidiosis being the most economically important parasitic disease ofchickens. Since 1949, preventive anticoccidials have been used but havenot been totally effective. Losses due to morbidity from coccidiosis,including reduced weight gains and egg production, and decreased feedconversion, persist. The cost of coccidiosis in broiler production hasbeen estimated at 1/2 to 1 cent per pound. Based on an annual productionof 3,000,000,000 broilers annually in the United States, losses wouldtotal between 60 and 120 million dollars. To this figure must be addedthe cost of anticoccidials estimated at 35 million dollars. Theseimpressive figures emphasize the importance of reducing the incidence ofcoccidiosis in chickens.

Of the nine genera of coccidia known to infect birds, the genus Eimeriacontains the most economically important species. Various species ofEimeria infect a wide range of hosts, including mammals, but ninespecies have been recognized as being pathogenic to varying degrees inchickens: Eimeria acervulina, E. mivati, E. mitis, E. praecox, E.hagani, E. necatrix, E. maxima, E. brunetti and E. tenella.

Although the Eimeria are highly host specific, their life cycles aresimilar. The developmental stages of the avian coccidia can beillustrated by the species Eimeria tenella, which proliferates in thececum of the chicken.

The life cycle of the Eimeria species begins when the host ingestspreviously sporulated oocysts during ground feeding or by inhalation ofdust. Mechanical and chemical action in the gizzard and intestinal tractof the chicken ruptures the sporulated oocyst, liberating eightsporozoites. The sporozoites are carried in the digestive contents andinfect various portions of the intestinal tract by penetration ofepithelial cells. Subsequent life stages involve asexual multiplefission, the infection of other epithelial cells, development ofgametes, and fertilization to produce a zygote which becomes an oocystwhich is passed out of the host with the droppings. The oocyst undergoesnuclear and cellular division resulting in the formation of sporozoites,with sporulation being dependent upon environmental conditions.Ingestion of the sporulated oocyst by a new host transmits the disease.

Of all species of Eimeria, E. tenella has received the most attention.E. tenella is an extremely pathogenic species, with death oftenoccurring on the fifth or sixth day of infection.

Before the use of chemotherapeutic agents, poultry producers' attemptsto control coccidiosis were limited to various management programs.These programs were directed toward attempts at sanitation throughdisinfection, or by mechanical removal of litter. Despite these efforts,sufficient oocysts usually remained to transmit the disease.

One means of combating the hazards of coccidia is immunization. Thismethod involves feeding to the poultry a small dose of oocysts of eachof the species of coccidia during the first weeks of life. However,dosage control has proven difficult as birds ingest daughter oocysts,with some birds developing severe coccidiosis and others remainingsusceptible. Also, since this procedure produces mixed infections,sometimes adequate immunity does not develop to all species given. Inaddition, immunity development is too slow for use with broilerproduction.

Another means of combating coccidia is drug treatment after the poultryis infected. One drug that has been used is sulfanilamide which hasshown anticoccidial activity against six species of coccidia. However,unless the drug treatment of the flock is quickly initiated afterdiagnosis of the disease, medication may be started too late to beeffective.

Ideally, the best method for combating coccidia is preventivemedication. Since the advent of the use of sulfonamide drugs, over fortycompounds have been marketed for preventive medication against coccidia.There have been many problems with the use of such drugs, includinganticoccidial contamination of layer flock feeds, inclusion of excessiveanticoccidial drugs in the feed causing toxicity in the birds andomission of the anticoccidial from the feed resulting in coccidiosisoutbreaks. A particularly frustrating problem has been the developmentof drug-resistant strains of coccidia. Moreover, there is a potentialfor drug residues being deposited in the meat.

Clearly, available methods for the control of coccidiosis have met withlimited success, and the need for a safe, efficient, and inexpensivemethod of combating avian coccidiosis remains.

The development of an effective anticoccidial vaccine is a desirablesolution to the problem of disease prevention. Vaccines produced bytraditional methods will require extensive development. There arereports of the production of attenuated strains through passage inembryos or cell culture. While this approach may eventually lead tosuccessful vaccines, not all the important species of Eimeria have beenadapted to growth in culture or embryos such that they are capable ofcompleting their life cycle.

Genetic engineering methodology presents the opportunity for analternative approach to vaccine development. It is known that genesencoding antigenic proteins of pathogenic organisms can be cloned intomicroorganisms. The antigenic proteins then can be expressed at highlevels, purified, and used as vaccines against the pathogenic organism.These antigenic proteins have the advantage of being non-infectious andare potentially inexpensive to produce. Such "subunit vaccines" havebeen prepared from antigen genes for a number of viruses such ashepatitis, herpes simplex and foot and mouth disease virus. An alternateapproach is to produce "synthetic vaccines", smallchemically-synthesized peptides, whose sequence is chosen based upon theamino acid sequence translation of vital antigen DNA. The advantages ofsuch "synthetic vaccines" over traditional vaccination with attenuatedor killed pathogenic organisms have been summarized by Lerner in Nature299:592-596 (1982).

It is now possible to produce foreign proteins, including eukaryoticproteins, in prokaryotic organisms such as gram positive or gramnegative bacteria. The process involves the insertion of DNA (derivedeither from enzymatic digestion of cellular DNA or by reversetranscription of mRNA) into an expression vector. Such expressionvectors are derived from either plasmids or bacteriophage and contain:(1) an origin of replication functional in a microbial host cell; (2)genes encoding selectable markers, and (3) regulatory sequencesincluding a promoter, operator, and a ribosome binding site which arefunctional in a microbial host cell and which direct the transcriptionand translation of foreign DNA inserted downstream from the regulatorysequences. To increase protein production and stability, eukaryoticproteins are often produced in prokaryotic cells as a fusion withsequences from the amino-terminus of a prokaryotic protein.β-Galactosidase or the product of one of the E. coli tryptophan operongenes have been used successfully in this manner. Expression vectorshave also been developed for expression of foreign proteins ineukaryotic host cells, e.g., yeast and chinese hamster ovary tissueculture cells.

Host cells transformed with expression vectors carrying foreign genesare grown in culture under conditions known to stimulate production ofthe foreign protein in the particular vector. Such host cell/expressionvector systems are often engineered so that expression of the foreignprotein may be regulated by chemical or temperature induction. Proteinswhich are secreted may be isolated from the growth media, whileintracellular proteins may be isolated by harvesting and lysing thecells and separating the intracellular components. In this manner, it ispossible to produce comparatively large amounts of proteins that areotherwise difficult to purify from native sources.

Such microbially produced proteins may be characterized by manywell-known methods, including the use of monoclonal antibodies,hereinafter referred to as "MAbs," which are homogeneous antibodies thatreact specifically with a single antigenic determinant and display aconstant affinity for that determinant, or by use of polyvalentantibodies derived from infected birds, which react with a variety ofdifferent antigens and often with multiple determinants on a singleantigen.

Alternate technology to the production of "sub-unit" or "synthetic"vaccines is the use of a fowl pox virus vector. The pox virus vacciniahas a long history of use as a vaccine and has been employed tovirtually irradicate smallpox in humans. It now has been demonstratedthat vaccinia virus can be effectively genetically engineered to expressforeign antigens (Smith et al., Nature 302:490-495 (1983); Panicali etal., Proc. Natl. Acad. Sci. USA 80:5364-5368 (1983); Mackett et al., J.of Virology 49:857-864 (1984)) and the engineered viruses can serve as alive vaccine against other viruses and infections besides smallpox. Fowlpox virus is very similar to vaccinia virus and many of the methodsdeveloped for vaccinia for the creation of recombinants expressingforeign antigens can be applied to fowl pox. Attenuated fowl pox virusengineered to produce arian coccidia antigens thus is another method toproduce an anti-coccidial vaccine. Live vaccines have the advantage ofbeing inexpensive to produce and are characterized by the production ofrapid immunity development.

A second type of live vaccine results in the presentation of antigen inthe gut where coccidia normally invades. This method utilizes secretionor outer surface expression of the antigen by harmless bacteriaintroduced into the intestinal microbial population by incorporation infeed. Secretion is obtained by fusion of an antigen gene to the genecoding for a protein which is normally secreted, leaving the necessarysecretion signal sequence intact. Outer surface expression is achievedby fusion of the antigen genes to the genes that code for proteinsnormally localized on the outer surface. (T. Silhavy, U.S. Pat. No.4,336,336.) This type of live vaccine is especially advantageous sincemanufacturing costs are minimal and the immune response stimulated is ofa type particularly effective against coccidia invasion of the gut.

SUMMARY OF THE INVENTION

This invention relates to novel recombinant antigenic proteins of aviancoccidiosis, and fragments thereof containing antigenic determinants,and to the genes that encode the antigenic peptides. It has now beenfound that particular polypeptides present in avian cells infected withcoccidiosis, when purified and isolated, contain an antigenicdeterminant or determinants which can elicit an antibody response. Thisinvention also relates to vaccines made using the novel antigenicproteins of avian coccidiosis and to methods of immunizing chickensagainst avian coccidia.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B shows the nucleotide sequence of the 5'-3' strand of cDNAencoding the E. acervulina antigen ac-1b gene.

FIGS. 2A-2B shows the amino acid sequence of E. acervulina antigenac-1b.

FIGS. 3A-3B shows the nucleotide sequence of the 5'-3' strand of cDNAencoding the E. acervulina antigen ac-6b gene.

FIGS. 4A-4B shows the amino acid sequence of E. acervulina antigenac-6b.

FIG. 5 shows the nucleotide sequence of the 5'-3' strand of cDNAencoding the E. tenella antigen tc-7a gene.

FIG. 6 shows the amino acid sequence of E. tenella antigen tc-7a.

FIG. 7 shows the nucleotide sequence of the 5'-3' strand of cDNAencoding the E. tenella clone tc-8a gene.

FIG. 8 shows the amino acid sequence of E. tenella clone tc-8a.

FIG. 9 shows the nucleotide sequence of the 5'-3' strand of cDNAencoding the E. tenella antigen tc-10a gene.

FIG. 10 shows the amino acid sequence of E. tenella antigen tc-10a.

As is well known in the art, due to the degeneracy of the genetic code,the DNA sequences given in the Figures for the genes and antigenicpeptides of this invention may be encoded by different DNA than thoserepresented. Thus, knowledge of an amino acid sequence does notnecessarily lead to a precise genetic sequence coding therefor. In allof the Figures with DNA and amino acid sequences the sequence is givenas the 5' to 3' strand. The abbreviations have the following standardmeanings:

A is deoxyadenyl

T is thymidyl

G is deoxyguanyl

C is deoxycytosyl

GLY is glycine

ALA is alanine

VAL is valine

LEU is leucine

ILE is isoleucine

SER is serine

THR is threonine

PHE is phenylalanine

TYR is tyrosine

TRP is tyryptophan

CYS is cysteine

MET is methionine

ASP is aspartic acid

GLU is glutamic acid

LYS is lysine

ARG is arginine

HIS is histidine

PRO is proline

GLN is glutamine

ASN is asparagine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to recombinant antigenic proteins, andfragments thereof containing antigenic determinants, that can elicit anantibody response against avian coccidiosis, and to the cloned genesthat encode the antigenic proteins and fragments. These antigenicproteins, and the fragments thereof containing antigenic determinants,will bind with a specific monoclonal antibody or with polyvalentantibodies from infected chickens, or from other animals that have beenimmunized with life forms of Eimeria or Eimeria proteins, directedagainst an antigenic protein of arian coccidia.

The antigenic proteins of this invention may be used for severalapplications: (1) the protein(s) can be used in an avian coccidia assayto detect antibodies against the coccidia; (2) antibodies may beprepared from the antigenic protein(s); (3) the protein(s) can be usedfor preparing vaccines against arian coccidiosis.

Antibodies directed against coccidial-antigens are used to identify, byimmunological methods, transformed cells containing DNA encodingcoccidial antigens. The MAbs are used as a tool for identifying cellscontaining DNA sequences encoding coccidial antigens that are eitherspecies specific or common to all nine species. Screening transformantswith polyvalent chicken antiserum is used to identify DNA sequencesencoding a wide spectrum of coccidial proteins which are antigenic inchickens upon infection. DNA sequences from the transformants thusidentified then may be incorporated into a microorganism for large scaleprotein production. The antigenic proteins, as native proteins or ashybrids with other proteins, may be used as vaccines to immunize birdsto protect them from subsequent infection.

In addition, the DNA sequences comprising the genes that encodeantigenic proteins and fragments thereof may be used as DNA probes. Theprobes have a variety of uses, including screening a DNA library foradditional genes that may encode antigenic determinants.

The DNA probe may be labeled with a detectable group. Such detectablegroup can be any material having a detectable physical or chemicalproperty. Such materials have been well-developed in the field ofimmunoassays and in general almost any label useful in such methods canbe applied to the present invention. Particularly useful areenzymatically active groups, such as enzymes (see Clin. Chem. 22:1243(1976)), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes(see U.S. Pat. Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (seeU.S. Pat. No. 4,134,792); fluorescers (see Clin. Chem. 25:353 (1979));chromophores; luminescers such as chemiluminescers and bioluminescers(see Clin. Chem. 25:512 (1979)); specifically bindable ligands; proximalinteracting pairs; and radioisotopes such as ³ H, ³⁵ S, ³² P, ¹²⁵ I and¹⁴ C Such labels and labeling pairs are detected on the basis of theirown physical properties (e.g., fluorescers, chromophores andradioisotopes) or their reactive or binding properties (e.g., enzymes,substrates, coenzymes and inhibitors). For example, a cofactor-labeledprobe can be detected by adding the enzyme for which the label is acofactor and a substrate for the enzyme. For example, one can use anenzyme which acts upon a substrate to generate a product with ameasurable physical property. Examples of the latter include, but arenot limited to, beta-galactosidase, alkaline phosphatase and peroxidase.

As used herein, the term "antigenic" or "antigenic determinant" is meantimmunologically cross-reactive antigenic determinants with which a givenantibody will react. Therefore, the antigenic peptides of this inventionwill include chemically synthesized peptides, peptides made byrecombinant DNA techniques, and antibodies or fragments thereof whichare anti-idiotypic towards the determinant of the peptides of thisinvention.

Several procedures may be used to construct a microorganism thatproduces an antigenic protein that binds with a monoclonal or polyvalentantibody that is directed against an antigenic protein of aviancoccidia. One such procedure can be divided into the following majorstages, each of which is described more fully herein: (1) recovery andisolation of messenger RNA (mRNA) found in organisms of the genusEimeria; (2) in vitro synthesis of complementary DNA (cDNA), usingcoccicidia mRNA as a template; (3) insertion of the cDNA into a suitableexpression vector and transformation of bacterial cells with thatvector; and, (4) recovery and isolation of the cloned gene or genefragment. This route is referred to as the mRNA route. The advantage tothis route is that only "expressed" genes are cloned, reducing thenumber of individual transformants required to represent the entirepopulation of genes.

An alternative procedure can be divided into the following major stageswhich will also be described more fully herein: (1) recovery andisolation of nuclear DNA found in organisms of the genus Eimeria; (2)fragmentation of the DNA and insertion into a suitable vector; (3)transformation into a suitable microbial host; (4) selection oftransformants containing a gene which specifies the antigen of interest;and, (5) recovery and isolation of the cloned gene or gene fragment.This route is referred to as the nuclear DNA route. The advantage tothis route is that all genes are cloned, allowing the identification ofgenes not expressed at the time from which mRNA is isolated. These mayinclude genes which are expressed during stages of the life cycle whichare not easy to isolate.

After recovery and isolation of the cloned gene that is derived from theprocedures discussed above, the cloned DNA sequence is advantageouslytransferred to a suitable expression vector/host cell system for largescale production of the antigenic protein.

The DNA sequence that is to be isolated encodes an antigenic proteinthat will elicit an immune response when administered to chickens whichwill protect them from subsequent infections. It is not necessary toisolate a complete coccidial gene encoding such a protein, since thoseportions of the protein termed antigenic determinants are sufficient fortriggering a protective immune response (Lerner, supra). This antigenicdeterminant should be on the surface of the folded microbially-producedprotein to trigger the response (Lerner, supra).

In the mRNA route, the sequence may be isolated from the sporozoite lifestage of the parasite. It has been demonstrated that part of theprotective immune response in chickens is directed against thesporozoite. Scientists at the U.S. Department of Agriculture detectedantibodies to sporozoite proteins in immune chicken serum, which alsoindicates that the sporozoite is a life stage that can be affected by animmune response in chickens. Antigenic proteins isolated from other lifestages also may be effective as vaccines.

MAbs or polyvalent antibodies which bind to various sporozoite proteinscan be used to identify cloned DNA sequences encoding those proteins.Such proteins can be isolated and used to elicit a protective immuneresponse in chickens.

Sporozoites can be obtained from oocysts by excystation using the methodof Doran and Vetterling, Proc. Helminthol Soc. Wash. 34:59-65 (1967),and purified by the leucopak filter technique of Bontemps and Yvore,Ann. Rech. Vet. 5:109-113 (1974). Although the method of Doran andVetterling has been found suitable for obtaining sporozoites fromoocysts, any method is suitable as long as the nucleic acids within thesporozoites remain intact. Also, sporozoite mRNA may be isolated fromintact sporulated oocysts, which contain the sporozoites.

mRNA Route

Isolation of mRNA coding for the antigenic proteins of interest isadvantageously accomplished by lysis of intact sporulated oocysts underconditions which minimize nuclease activity. This is accomplished usinga modification of the procedure described by Pasternak et al., Molec.Biochem. Parisitol. 3:133-142 (1982). Total RNA may be isolated bygrinding the oocysts with glass beads in a solution containing sodiumdodecyl sulfate (SDS) and proteinase K. After denaturation anddegradation of oocyst proteins, the RNA is isolated by extraction of thesolution with phenol and precipitation with ethanol. Oligo(dT)-cellulose chromatography then can be used to isolate mRNA from thetotal RNA population.

Proteins coded for by the isolated mRNA can be synthesized, in vitro,using a cell-free translation system. A number of cell-free translationsystems have been devised, such as wheat germ extract (Martial et al.,Proc. Nat'l Acad. Sci. USA 74:1816-1820 (1977)), rabbit reticulocytelysate (Pelham and Jackson, Eur. J. Biochem. 67:247-256 (1976)), andoocytes from Xenopus laevis (Sloma et al., Methods in Enzymology79:68-71 (1981)). The rabbit reticulocyte lysate is preferred for thetesting of sporozoite mRNA. The rabbit reticulocyte lysate can besupplemented with a radioactively labeled amino acid, such as [³⁵S]-methionine, so that the resulting proteins contain a tracer. Thevarious protein products may be reacted with polyvalent chicken antiseraor MAbs previously described, followed by reaction with goatanti-chicken IgG in the case of the polyvalent antibodies andStaphylococcus aureus Protein A, or in the case of the MAbs, justProtein A. Protein A binds any of the mouse or goat antibodies to forman immunoprecipitated complex. The products of the translation and ofthe immunoprecipitation are visualized by gel electrophoresis followedby fluorography. The mRNA fractions found to produce proteins that reactwith the antisera in this system are used for ds-cDNA synthesis.Alternatively, to avoid missing any antigens which are not synthesizedefficiently, in vitro, or are not immunoprecipitated efficiently, totalmRNA is used for cDNA synthesis.

Synthesis of cDNA employs arian myeloblastosis virus reversetranscriptase. This enzyme catalyzes the synthesis of a single strand ofDNA from deoxynucleoside tri-phosphates on the mRNA template. (Kacianand Myers, Proc. Nat'l Acad. Sci. USA 73:2191-2195 (1976).) The polyr(A) tail of mRNA permits oligo(dT) (of about 12-18 nucleotides) to beused as a primer for cDNA synthesis. The use of a radioactively labeleddeoxynucleoside triphosphate facilitates monitoring of the synthesisreaction. Generally, a ³² p-containing deoxynucleoside triphosphate,such as [α-³² P]dCTP, may be used advantageously for this purpose. ThecDNA synthesis is generally conducted by incubating a solution of themRNA, the deoxynucleoside triphosphates, the oligo(dT) 12-18 and reversetranscriptase for 10 minutes at 46° C. The solution also preferablycontains small amounts of actinomycin D and dithiothreitol to promotefull length synthesis. (Kacian and Myers, supra.) After incubation,ethylenediaminetetraacetic acid (EDTA) is added to the solution, and thesolution is extracted with phenol:chloroform. The aqueous phase isadvantageously purified by gel filtration chromatography, and thecDNA-mRNA complex in the eluate is precipitated with alcohol.

The mRNA can be selectively hydrolyzed in the presence of the cDNA withdilute sodium hydroxide at an elevated temperature. Neutralization ofthe alkaline solution and alcohol precipitation yields a single-strandedcDNA copy.

The single-stranded cDNA copy has been shown to have a 5' poly (dT)tail, and to have a 3' terminal hairpin structure, which provides ashort segment of duplex DNA. (Efstratiadis et al., Cell 7:279-288(1976)). This 3' hairpin structure can act as a primer for the synthesisof a second DNA strand. Synthesis of this second strand is conductedunder essentially the same conditions as the synthesis of the cDNA copy,except that the Klenow fragment of E. coli DNA polymerase I (Klenow etal., Eur. J. Biochem. 22:371-381 (1971)) is substituted for reversetranscriptase. The duplex cDNA recovered by this procedure has a 3'loop, resulting from the 3' hairpin structure of the single-strandedcDNA copy. This 3' loop can be cleaved by digestion with the enzyme, S1nuclease, using essentially the procedure of Ullrich et al., Science196:1313-1319 (1977). The S1 nuclease digest may be extracted withphenol-chloroform, and the resulting ds-cDNA precipitated from theaqueous phase with alcohol.

For purposes of amplification and selection, the ds-cDNA prepared asdescribed above is generally inserted into a suitable cloning vector,which is used for transforming appropriate host cells. Suitable cloningvectors include various plasmids and phages, but a bacteriophage lambdais preferred.

For a cloning vector to be useful for the expression of foreign proteinswhich are to be detected with antibodies, it should have several usefulproperties. Most importantly, it should have a cloning site within agene which is expressed in the host being used. There should also be ameans of controlling expression of the gene. The vector should be ableto accept DNA of the size required for synthesis of the desired proteinproduct and replicate normally. It is also useful to have a selectableproperty which allows identification of vectors carrying inserts. Acloning vector having such properties is the bacteriophage λgt11 (ATCC37194) (Young and Davis, Proc. Nat'l Acad. Sci. USA 80:1194-1198(1983)). This vector has a unique EcoRI site near the end of thebacterial gene coding for β-galactosidase. That site can be used forinsertion of foreign DNA to make hybrid proteins made up ofβ-galactosidase and the foreign gene product. The expression ofβ-galactosidase is under control of the lac promoter and can be inducedby the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). The λgt11phage contains 43.7 kb of DNA which is considerably smaller than wildtype λ. This allows insertion of pieces of DNA up to 8.3 kb in length,before the DNA becomes too large to fit inside the phage head. BecauseDNA is inserted into the gene for β-galactosidase, transformants havinginserts can easily be distinguished from those which do not by lookingfor β-galactosidase activity. An indicator dye,5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal), can be incorporatedwith agar plates. β-galactosidase cleaves this molecule to give a blueproduct, thus allowing examination of the cultures for the presence ofactive β-galactosidase. Those plaques having inserts are colorless onX-gal plates because the insertion of foreign DNA into theβ-galactosidase gene has eliminated its activity.

The ds-cDNA can be conveniently inserted into the phage by addition ofEcoRI linkers to the DNA and ligation into the EcoRI-cut λgt11 DNA.After ligation of the cDNA into the phage DNA, the DNA is packaged, invitro, into λ phage heads (Enquist and Sternberg, Methods in Enzymology68:281-298 (1979) and those phages are used to infect a suitableλ-sensitive host. With the proper choice of host, the phage may bescreened as plaques or lysogens (colonies).

Aside from the E. coli/bacteriophage λgt11 system described, many otherhost/vector combinations have been used successfully for the cloning offoreign genes in E. coli (Principles of Gene Manipulation, 2nd Ed., Oldand Primrose, Univ. of California Press, 32-35, 46-47 (1981)) including"open reading frame" vectors, described in detail later.

The foregoing discussion has focused on cloning procedures in gramnegative bacteria, e.g., E. coli. Alternatively, foreign genes may becloned into plasmid vectors that will transform and replicate in a grampositive bacterium such as Bacillus subtilis (Old and Primrose, supra,pp. 51-53) or in a eukaryotic host cell such as yeast (Old and Primrose,supra, pp. 62-68) filamentous fungi, insect cells (U.S. Pat. No.4,745,051 and U.S. Pat. No. 4,879,236) and mammalian cells. Cloningvectors have been constructed which transform both yeast and E. coli.Such vectors are termed "shuttle vectors" and may be transferred, alongwith the cDNA they carry, between the two host microorganisms (Storms,et al., Journal of Bacteriology 140:73-82 (1979); and Blanc et al.,Molec. Gen. Genet. 176:335-342 (1979). Shuttle vectors also exist whichreplicate in (and may carry cloned genes into) both E. coli and B.subtilis (Old and Primrose, supra, at p. 53). Vectors derived from theother bacteriophages such as M13 have also proven useful in the cloningof foreign genes (Old and Primrose, supra, Chap. 5). Any of thesetechniques can be employed, if desired, in the constructions of thepresent invention.

The DNA described herein may be inserted into the above vectors byvarious techniques including homopolymeric tailing, blunt-end ligationor by use of linker molecules (Old and Primrose, supra, at p. 92).

Many immunological methods for screening clone banks for thoseexpressing a desired protein are known and include procedures describedby Engvall and Pearlman, Immunochemistry 8:871-874 (1971); Koenen etal., The European Molecular Biology Organization Journal, Vol. 1, No. 4,pp. 509-512 (1982); Broome et al., Proc. Natl. Acad. Sci., USA75:2746-2749 (1978); Villa-Komaroff et al., Proc. Natl. Acad. Sci., USA75:3727-3731 (1978); Anderson et al., Methods in Enzymology 68:428-436(1979); Clarke et al., Methods in Enzymology 68:436-442 (1979); Ehrlichet al., Methods in Enzymology 68:443-453 (1979); Kemp et al., Proc.Natl. Acad. Sci., USA 78:4520-4524 (1981).

By the cloning procedures outlined, thousands of recombinantbacteriophage are generated. In order to screen them for production ofcoccidial antigens, two antibody screens can be utilized. Both screeningmethods depend upon expression of the coccidial antigenic protein eitheralone or as a fusion protein with a bacterial gene. In the examplesincluded herein, the coccidial antigens are produced as fusions with E.coli β-galactosidase. The screening methods, therefore, depend onexpression of the fusion product and detection of the product byreaction with antibodies, either monoclonal or polyvalent, directedagainst that antigen.

The recombinant bacteriophages can be used to infect a suitable E. colihost which allows the formation of phage plaques on agar (or agarose)plates. The plaques can be transferred to nitrocellulose membranes whilebeing induced with IPTG. The proper antibodies are then reacted with thefilters. After reaction of the primary antibodies with the filters, thepositive reactions are detected by reaction with either [¹²⁵ I]Staphylococcus aureus Protein A or a second antibody conjugated withhorse-radish peroxidase.

Alternatively, the recombinant bacteriophages can be used to infect anE. coli host in which lysogens are produced at a high frequency. In thiscase, the transformants can be screened as colonies. The colonies aregrown on a cellulose acetate filter under non-induced conditions. Afterthe colonies have reached a suitable size, the cellulose acetate filteris placed over a nitrocellulose filter which is on an agar platecontaining IPTG. The colonies are incubated at elevated temperatures toinduce phage production, while expression of the β-galactosidase gene isinduced by inclusion of IPTG. After a suitable incubation period, duringwhich some lysis of the colonies occurs with release of proteins throughthe cellulose acetate filter onto the nitrocellulose filter, thecellulose acetate filter is removed. The nitrocellulose filter isprocessed as described above for screening of plaques.

The phages giving positive signals in the antibody-screening procedurecan be shown to contain sequences coding for coccidial proteins byexcision of the DNA originally inserted into the phage DNA andexamination of the ability of that DNA to hybridize with coccidia mRNAor coccidia genomic DNA. The nucleotide sequence of the cDNA insert isdetermined using the methods of Sanger et al., Proc. Natl. Acad. Sci.,USA 74:5463-5467 (1977); or Maxam and Gilbert, Proc. Natl. Acad. Sci.,USA 74:560-S64 (1977).

Nuclear DNA Route

Another method of cloning coccidial antigens begins with isolation ofnuclear DNA from oocysts. This DNA is then broken into fragments of asize suitable for insertion into a cloning vector. To obtain suchfragments, one can use mechanical shearing methods such as sonication orhigh-speed stirring in a blender to produce random breaks in the DNA.Intense sonication with ultrasound can reduce the fragment length toabout 300 nucleotide pairs. (Old and Primrose, supra, p. 20.)Alternatively, nuclear DNA may be partially digested with DNAseI, whichgives random fragments, with restriction endonucleases, which cut atspecific sites, or with mung bean nuclease in the presence of formamide,which has been shown with some related organisms (McCutchan, T. F., etal. Science 225:625-628 (1984)) to produce DNA fragments containingintact genes.

These nuclear DNA fragments may be inserted into any of the cloningvectors listed for the cloning of cDNA in the mRNA experimental method.If the nuclear DNA is digested with a restriction endonuclease, it canbe inserted conveniently into a cloning vector digested with the sameenzyme, provided the vector has only one recognition site for thatenzyme. Otherwise, DNA fragments may be inserted into appropriatecloning vectors by homopolymeric tailing or by using linker molecules(Old and Primrose, supra, at p. 92).

Advantageously, the nuclear DNA fragments are cloned into "open readingframe" vectors which are designed to simultaneously clone and expressforeign genes or fragments thereof. Several such vectors are known inthe art, including those described by Weinstock et al., Proc. Natl.Acad. Sci., USA 80:4432-4436 (1983); Keonen et al., The EuropeanMolecular Biology Organization Journal 1, 4, pp. 509-512 (1982); Rutheret al., 79:6852-6855 (1982); Young and Davis, supra; and Gray et al.,Proc. Natl. Acad. Sci., USA 79:6598-6602 (1982).

Open reading frame (ORF) vectors have been used to clone bothprokaryotic and eukaryotic genomic DNA or cDNA. These vectors generallycontain a bacterial promoter operably linked to an amino terminalfragment of a prokaryotic gene. A carboxy terminal fragment of a genewhich encodes a product for which an assay is known (e.g., the E. colilacZ gene which encodes β-galactosidase) is located downstream. Thesequences between the amino terminal gene fragment and the lacZ fragmentinclude restriction endonuclease recognition sites useful for insertionof foreign genes and, in some cases, also place the lacZ fragment out ofreading frame for translation with respect to the amino terminal genefragment. When foreign DNA is inserted into these vectors (by blunt endligation, homopolymeric tailing, ligation of cohesive termini, or theuse of linkers), a certain percentage of recombinants will have receivedforeign DNA of a length that puts the lacZ gene in phase with thereading frame set by the amino terminal gene fragment. The result isproduction of a "tribrid" protein comprising the polypeptides encoded bythe amino terminal gene fragment, the cloned DNA, and the lacZ gene.Such recombinants are identified on MacConkey agar plates or on agarplates containing "Xgal" (5-bromo-4-chloro-3-indolyl-β-D galactoside)because the β-galactosidase activity of the tribrid protein cleaves thedye in such plates, turning colonies red (MacConkey agar) or blue(Xgal). β-galactosidase can carry a wide range of protein sequences atits amino terminus and still retain biological activity. Alternatively,the insert may be inserted to inactivate a gene by interrupting thesequence. The insert may be in the correct reading frame to produce ahybrid gene consisting of the amino-terminus of the bacterial gene andsequences from the insert gene at the carboxy terminus.

Only recombinants receiving exons (i.e., coding sequences of genes,which have no stop codons) which are in-frame with respect to the aminoterminal gene fragment are detected by this method. ORF vectors areuseful for cloning genes for which no DNA or protein sequence dataexists, if antibodies against the gene product exist. Screening of theclone bank may be accomplished by immunological methods which make RNAor DNA hybridization probes unnecessary. The immunological screeningmethods mentioned for the mRNA route can be used.

Plasmid DNA is isolated from transformants found to be "positive" by theabove screening methods. The nuclear DNA inserts of these plasmids arethen subjected to DNA sequencing. Once the nucleotide sequence is known,it is possible by known methods to chemically synthesize all or part ofthe cloned coccidial genes. The synthesis of fragments of the clonedgenes, followed by insertion of the gene fragments into expressionvectors as described below and reaction of the polypeptides producedwith MAbs allows detection of those portions of the gene which areantigenic determinants.

Once a cloned DNA sequence is identified as encoding a protein thatbinds antibodies directed against coccidial proteins, it may betransferred to expression vectors engineered for high-level productionof the desired antigenic protein. The expression vectors are transformedinto suitable host cells for production of the antigenic protein. Thesehost cells may include both prokaryotic and eukaryotic organisms. Theprokaryotic host cells include E. coli and B. subtilis. The eukaryotichost cells include yeast, insect cells, and mammalian cells.

Coccidial antigens advantageously may be produced at high levels in E.coli as a fusion protein comprising the antigen and an amino terminalportion of the β-subunit of the enzyme tryptophan synthetase (theproduct of the E. coli trpB gene). This fusion is accomplished byinserting a DNA sequence encoding a coccidial antigen into a plasmidvector carrying the trpB gene.

The expression vector used may be one in which expression of the fusionantigenic protein is highly regulatable, e.g., by chemical induction ortemperature changes. An expression vector with such regulatorycapability is the plasmid pGX2606, which contains a hybrid λO_(L) P_(R)regulatory region as described in copending application Ser. No. 534,982filed Sep. 23, 1983. Host expression vector systems in which expressionof foreign proteins is regulatable have the advantage of avoidingpossible adverse effects of foreign protein accumulation as high celldensities are reached. Some investigators have proposed that expressionof gene fragments such as those encoding antigenic determinants mayavoid the deleterious effects that expression of the entire antigenicprotein would have on E. coli host cells. (Helfman et al., Proc. Natl.Acad. Sci., USA 80:31-35 (1983)).

Coccidial antigens also may be produced in high levels as fusions at thecarboxy-terminal of E. coli β-galactosidase, as they are directlyobtained by use of the cloning vector λgt11. The fusedβ-galactosidase-coccidia antigen gene is transferred with all of theassociated regulatory elements to a small plasmid, where synthesis ofthe gene product is regulated by the lac promoter, which is transferredalong with the fusion gene from the phage to the plasmid. Such a smallplasmid is PGX1066 (plasmid pGX1066 is present in E. coli strain GX1186,ATCC 39955) which carries the gene for ampicillin resistance and has abank of restriction sites which are useful for insertion of DNAfragments. Synthesis of the fusion protein is induced by addition ofIPTG, the inducer of the lac operon.

An effective subunit vaccine against avian coccidiosis may consist of amixture of antigen proteins derived from several species of Eimeria.Alternatively, production costs may be decreased by producing two ormore antigen proteins as one fusion protein thus reducing the requirednumber of fermentations and purifications. Such a fusion protein wouldcontain the amino acid sequence comprising an antigenic epitope of eachantigen protein (or repetitions of those sequences) with variableamounts of surrounding nonantigenic sequence. A hybrid gene designed tocode for such a protein in E. coli would contain bacterial regulatorysequence (promoter/operator) and the 5' end of an E. coli gene (theribosome binding site and codons for several amino acids) to ensureefficient translation initiation followed by the coding sequences forthe antigenic epitopes all fused in the same reading frame.

E. coli cells transformed with the expression vector carrying a clonedcoccidial antigen sequence are grown under conditions that promoteexpression of the antigenic polypeptide. The antigenic protein is thenpurified from the cells and tested for ability to elicit an immuneresponse in chickens that will protect them from subsequent Eimeriainfections. The purified protein may be used to immunize the birds. Thepurified protein may be combined with suitable carriers and adjuvantsand administered to birds in their feed or by injection. Alternatively,live microorganisms containing the DNA sequences encoding the coccidialantigens may be fed to chickens. Such microorganism are advantageouslythose which normally inhabit the avian intestinal tract, such as E. colior coryneform bacteria.

In a preferred system, the microorganisms are transformed with anexpression vector in which the sequences encoding the coccidial antigenare fused in frame to a gene or gene fragment encoding a host cell outermembrane protein or secreted protein, such as the E. coli lamb protein,the λ receptor. The antigenic protein is therefore continuouslypresented in the host at the location of infection by the parasites. Itis known that foreign proteins fused in expression vectors to outermembrane or secreted proteins have been presented at the cell surface orsecreted from their host cells. (Weinstock, supra, and Silhavy, U.S.Pat. No. 4,336,336 which is herein incorporated by reference.)

In another preferred system for development of live vaccines, anattenuated fowl pox virus expression vector is utilized. Fowl pox hasthe capacity to accommodate several coccidia genes allowing theproduction of multivalent vaccines. Currently, attenuated fowl pox virusis utilized as a vaccine to protect commercial flocks against fowl poxinfection. Virus preparation and treatment of birds with fowl pox virusgenetically engineered to produce coccidia antigens is the same as theconventional methods of pox vaccine use currently practiced.

Pox viruses are among the most complex viruses known with very highmolecular weight double-stranded DNA genomes. With the most studied poxvirus, vaccinia, it has been demonstrated that the pox genome can easilyaccommodate inserts of foreign DNA capable of coding for foreignantigenic proteins (Smith et al., supra; Panicali et al., supra; Mackettet al., supra). When a foreign gene is incorporated into the pox virusgenome under the control of a pox promoter regulatory sequence, theforeign antigen is expressed upon infection in the cytoplasm of the cellwhere the pox virus replicates. Successful insertion and expression ofcoccidia antigen genes within the fowl pox genome is dependent uponidentifying a nonessential region of the pox DNA for antigen geneinsertion and ensuring an active pox promoter is situated 5' of thedesired coccidia gene.

Insertion of DNA into the pox genome is accomplished by in vivorecombination. Pox DNA is not infectious presumably because itscytoplasmic location requires the presence of pox virus specific RNA andDNA polymerases that are normally carried into the cell by the virion.DNA sequence information from vaccinia virus (Weir and Moss, J. ofVirology 46:530-537 (1983); Venkatesan et al., Cell 125:805-813 (1981))demonstrates sequence patterns in regulatory regions that are likely tobe unique to vaccinia genes and thus not recognized by cellular enzymes.Because the pox DNA is not infectious, foreign DNA insertion into thefowl pox genome is accomplished by in vivo recombination as has beendemonstrated with vaccinia to occur at high frequency (Weir et al.,Proc. Natl. Acad. Sci., USA 79:1210-1214 (1982)). A fowl pox virusinfection of chick embryo fibroblasts is followed by transfection usingthe CaCl₂ precipitation technique (Graham et al., Virology 52:456-457(1973); Stow et al., J. Virology 28:6182-192 (1978)) with plasmid DNAthat includes the coccidia antigen gene placed under the control of apromoter functional in fowl pox, and DNA sequence homology with fowlpox. During the course of the infection recombination occurs. If acoccidia DNA sequence is inserted within the fowl pox homologoussequence on the transfected plasmid, upon recombination the coccidia DNAsequence is, in some cases, inserted into the pox virus genome. Theinfected cells and virus from a recombination attempt are harvested andfresh chick embryo fibroblast cells grown as a monolayer in tissueculture are infected at a low multiplicity such that individual plaquesresulting from an initial single virus infection can be identified usingconventional techniques. Desired recombinant viruses are identifiedusing an in situ hybridization technique (Villarreal and Berg, Science196:183-185 (1977)) using radioactive coccidia DNA sequence as probe.Alternatively, vital DNA immobilized on nitrocellulose paper preparedfrom cells infected by plaque purified virus or cells infected withpools of potential recombinant viruses can be used for identification ofdesired recombinant viruses. Immunological screening of fixed cells(Gremer et al., Science 228:737-740 (1985)) is an alternative tohybridization.

The region of fowl pox DNA included in the plasmid vector must be from anonessential region, and is chosen by randomly testing segments of fowlpox DNA for regions that allow recombinant formation without seriouslyaffecting virus viability using the method described above. Fowl pox DNAis purified (Muller et al., J. Gen. Virology 38:135 (1977); Gafford etal., Virology 89:229 (1978)), randomly sheared to about 3 kilobases andcloned into a small bacterial plasmid, such as pGx1066, creating severaldifferent isolates. Foreign DNA must be inserted into the fowl poxportion of the plasmids before testing the effect of recombination uponvirus viability. To accomplish this, E. coli transposon insertions suchas λδ (Guyer, Methods in Enzymology 101:362-369 (1983)) can be readilyplaced within the fowl pox portion of the plasmid. Cotransfections thatresult in viable fowl pox recombinants containing λδ sequence identifydesirable nonessential fowl pox DNA for use in cotransfection plasmids.

Fowl pox DNA regions with partial sequence homology to the thymidinekinase gene of vaccinia identified by hybridization experiments are alsouseful for inclusion in the cotransfection plasmid since the thymidinekinase gene of vaccinia has been shown to be nonessential (Weir (1982),supra; Mackett et al., Proc. Natl. Acad. Sci., USA 79:4927-4931 (1982);Hruby and Ball, J. Virology 43:403-408 (1982)).

Placement of the coccidia antigen gene under the control of a fowl poxpromoter is carried out by conventional in vitro manipulation of theplasmid before concurrent transfection and fowl pox infection. Promotersequences useful for driving expression of the coccidia antigens couldbe identified by determination of the DNA sequence located 5' to fowlpox genes. Promoter sequences are then synthesized chemically andincluded in the plasmid vector adjacent to endonuclease cloning siteswithin the fowl pox homologous region of the plasmid. Putative promotersequences identified through DNA sequencing of vaccinia DNA (Venkatesanet al. (1981), supra; Weir and Moss (1983), supra) are also chemicallysynthesized and compared with fowl pox promoters for optimal effect.Putative fowl pox promoters are verified by cloning them 5' of a testgene with an easily measured translation product such as chloramphenicolacetyl-transferase (Gorman et al., Molecular and Cellular Biology2:1044-1057 (1982)) in a bacterial plasmid. The plasmid is used totransfect fowl pox infected tissue culture cells and the cells areassayed for transient expression of the test gene.

Vaccinia virus has a broad host range and does infect chickens. Thus thevectors and methods already developed for vaccinia could be utilized todevelop vaccines for avian coccidia and coccidiosis in any other genusincluded in the vaccinia host range. This approach requires cautionsince vaccinia is severely pathogenic to a small proportion of the humanpopulation.

A good alternative to pox vectors would be to utilize a herpes virussuch as Marek's Disease virus or Herpes virus of turkeys. Attenuatedforms of both viruses are currently used as live vaccines to preventMarek's disease in poultry. Similar to pox viruses, herpes viruses havelarge double stranded DNA genomes and are good candidates for geneticengineering using in vivo recombination methods similar to thosedeveloped for vaccinia. The advantage of engineering Marek's diseasevirus to also provide protection against coccidia infection is thatcoccidia protection is provided at no additional production cost abovethe Marek's Disease Vaccine that is already generally in Use.

The production of coccidia antigen by fowl pox recombinants is verifiedby immunological analysis of the protein produced in chick embryofibroblast tissue culture cells after infection and also by testing thecirculating antibody of birds infected with recombinant fowl pox virusfor cross reaction with whole coccidia or protein isolated from coccidiaof the appropriate species.

The cloned antigenic proteins used in vaccines above are tested fortheir ability to elicit an immune response in chickens that protects thebirds from subsequent infection by any of the important species ofEimeria, including E. tenella, E. acervulina, E. brunetti, E. mivati, E.maxima, E. praecox, E. mitis, and E. necatrix. The cloning proceduresdescribed above may be repeated until DNA sequences encoding coccidialantigens that collectively protect chickens against coccidiosis areisolated and used as a vaccine by the methods above.

In addition to cloned antigenic proteins which may be useful as vaccinesto protect against coccidiosis, another useful alternative which may bederived from cloning antigen genes is the use of small, syntheticpeptides in vaccines (see Lerner, supra). After the sequence ofantigenic proteins is determined, it is possible to make syntheticpeptides based on that sequence. The peptides are conjugated to acarrier protein such as hemocyanin or serum albumin and the conjugatethen can be used to immunize against coccidia.

It is contemplated that the procedures described may also be used toisolate antigenic proteins from other coccidia species that can be usedin vaccines to protect other domestic animals from coccidiosis.

The following examples are supplied in order to illustrate, but notlimit, the present invention.

EXAMPLE 1 Identification of an Eimeria Acervulina cDNA Clone EncodingAntigen ac-Ib with a Monoclonal Antibody

In order to identify antigens of Eimeria acervulina, expressionlibraries were prepared in lambda vector, λgt11, using cDNA preparedfrom polyA mRNA isolated from E. acervulina oocysts. The methods usedfor construction of the libraries is described in Genex PatentApplication, PCT/US89/02918, incorporated herein by reference. The cDNAexpression library was screened with monoclonal antibody (MCA) 12-09which was raised against the sporozoite stage of E. acervulina. The MCAwas obtained from Dr. Harry Danforth, U.S. Department of Agriculture,Beltsville, Md.

The library to be screened was plated on a host that allows lysis andplaque formation. During induction of the antigens encoded by the phage,the plaques were transferred to nitrocellulose filters. Phage thatproduce antigens cross-reactive with MCA 12-09 were identified byscreening the filters with MCA 12-09. The cDNA inserts from the MCA12-09 positive phage were cloned into bacteriophage M13 and subjected tosequence analysis. Following sequence analysis, E. acervulina antigenac-1b was identified. The complete sequence of ac-1b and its 25.2 Kdtranslation product are given in FIGS. 1A-1B and 2A-2B.

Antigens ac-1b was expressed in E. coli after insertion into the plasmidexpression vector pEX-32b. Antigen ac-1b is encoded in expression vectorpGX5361 and is expressed as a fusion protein with 11 Kd of the MS-2polymerase under control of the P_(L) promoter. The host strain pop2136contains a temperature sensitive repressor of P_(L) and expression ofthe fusion protein is fully induced at 42° C.

EXAMPLE 2 Identification of an Eimeria Acervulina cDNA Clone EncodingAntigen ac-6b with a Monoclonal Antibody

Using the techniques described in Example 1 E. acervulina libraries werescreened with MCA 12-09. Phage that produce antigens crossreactive withthe monoclonal were identified. The cDNA inserts from the MCA 12-09positive phage were cloned into bacteriophage M13 and subjected tosequence analysis. Following sequence analysis, E. acervulina antigenac-6b was identified.

Antigen ac-6b showed sequence homology to the carboxy terminal sequenceof E. tenella antigen GX3262, which has been shown to be protectiveagainst Eimeria infections in chickens (Miller et al., Infect. Immun.57:2014-2020 (1989), Danforth et al., Poultry Sci. 68:1643-1652 (1989).In-order to recover the entire ac-6b gene, an E. acervulina cDNAexpression library was screened with an 18 bp oligonucleotidecorresponding to the extreme 5'-sequence of the original ac-6b sequence.A plaque that hybridized to the oligonucleotide was identified and shownby sequence analysis to contain the complete ac-6b gene which encodes a21.6 Kd coccidial peptide. The sequence of ac-6b and its translationproduct is given in FIGS. 3A-3B and 4B-4B.

Antigen ac-6b was expressed in E. coli after insertion into the plasmidexpression vector pEX-32b. Antigen ac-6b is encoded in expression vectorpGX5368 and is expressed as a fusion protein with 11 Kd of the MS-2polymerase under control of the P_(L) promoter. The host strain pop2136contains a temperature sensitive repressor of P_(L) and expression ofthe fusion protein is fully induced at 42° C.

EXAMPLE 3 Identification of an Eimeria Tenella cDNA Clone EncodingAntigen tc-7a with a Monoclonal Antibody

In order to identify antigens of Eimeria tenella, expression librarieswere prepared in the lambda vector, λgt11, using cDNA prepared frompolyA mRNA isolated from E. tenella oocysts. The cDNA expression librarywas screened with monoclonal antibody (MCA) 12-07 which was raisedagainst the sporozoite stage of E. tenella. The MCA was obtained fromDr. Harry Danforth, U.S. Department of Agriculture, Beltsville, Md.

The library to be screened was plated on a host that allows lysis andplaque formation. During induction of the antigens encoded by the phage,the plaques were transfer to nitrocellulose filters. Phage that produceantigens cross-reactive with MCA 12-07 were identified by screening thefilters with MCA 12-07. The cDNA inserts from the MCA 12-07 positivephage were cloned into bacteriophage M13 and subjected to sequenceanalysis. Following sequence analysis, E. tenella antigen tc-7a wasidentified. Antigen tc-7a consists of an open reading frame of 540 bpthat encodes the carboxy terminal fragment of the E. tenella protein.The DNA sequence of tc-7a and its 16.6 Kd translation product are shownin FIGS. 5 and 6.

Antigen tc-7a was expressed in E. coli after insertion into the plasmidexpression vector pEX-32b. Antigen tc-7a is encoded in expression vectorpGX5391 and is expressed as a fusion protein with 11 Kd of the MS-2polymerase under control of the P_(L) promoter. The host strain pop2136contains a temperature sensitive repressor of P_(L) and expression ofthe fusion protein is fully induced at 42° C.

EXAMPLE 4 Identification of an Eimeria Tenella cDNA Clone EncodingAntigen tc-8a with a Monoclonal Antibody

As described in Example 3, E. tenella libraries were screened with MCA12-07. Phage that produce antigens crossreactive with the monoclonalwere identified. The cDNA inserts from the MCA 12-07 positive phage werecloned into bacteriophage M13 and subjected to sequence analysis.Following sequence analysis E. tenella antigen tc-8a was identified.

Antigens tc-8a is encoded on a short DNA fragments of 117 bp. Thefragment consists of an open reading frame covering its entire length.The DNA sequence of the fragment and its 3.5 Kd translation product isshown is FIGS. 7 and 8.

EXAMPLE 5 Identification of an Eimeria tenella cDNA Clone EncodingAntigen tc-10a with a Monoclonal Antibody

As described in Example 3, E. tenella libraries were screened with MCA12-07. Phage that produce antigens cross-reactive with the monoclonalwere identified. The cDNA inserts from the MCA 12-07 positive phage werecloned into bacteriophage M13 and subjected to sequence analysis.Following sequence analysis, E. tenella antigen tc-10a was identified.

Antigens tc-10a is encoded on a short DNA fragments of 228 bp. Thefragment consists of an open reading frame covering its entire length.The DNA sequence of the fragment and its 7.8 Kd translation product isshown is FIGS. 9 and 10.

While certain representative embodiments and details have been shown forthe purpose of illustrating the invention, it will be apparent to thoseskilled in the art that various changes and modifications may be madetherein without departing from the scope of the invention.

What is claimed is:
 1. An antigenic composition capable of eliciting anantibody response against arian coccidiosis, wherein said antigeniccomposition comprises an isolated polypeptide having an amine acidsequence selected from the group consisting of the amine acid sequencesshown in FIG. 2A-2B, FIG. 8, and FIG.
 10. 2. A method for reducing theseverity of avian coccidiosis comprising administering to a bird aneffective amount of an antigenic composition comprising an isolatedpolypeptide having an amine acid sequence selected from the groupconsisting of the amine acid sequences shown in FIG. 2A-2B, FIG. 4A-4B,FIG. 6, FIG. 8, and FIG. 10.